benzylideneimidazolinone

8/2/2019 benzylideneimidazolinone

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Published: November 9, 2010

r 2010 American Chemical Society 32 dx.doi.org/10.1021/co100012k| ACS Comb. Sci. 2011, 13, 3238

RESEARCH ARTICLE

pubs.acs.org/acscombsci

Fluorescence Response Profiling for Small Molecule Sensors Utilizingthe Green Fluorescent Protein Chromophore and Its Derivatives

Jun-Seok Lee,,#,3Anthony Baldridge, ,3 Suihan Feng,, Yang SiQiang, )Yun Kyung Kim,,#

Laren M. Tolbert,*,^ and Young-Tae Chang*,,, )

Department of Chemistry, MedChem Program of Life Sciences Institute, National University of Singapore, 3 Science Drive 3,Singapore 117543, Singapore)Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research

(A*STAR), Biopolis 138667, Singapore^School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, United States#Life/Health Division, Integrated Omics Center, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea

bS Supporting Information

ABSTRACT: Using afl

uorescence response profi

le, a systematicexamination was performed for synthetic chromophores of thegreen fluorescent protein (GFP) to discover new small moleculesensors. A group of 41 benzylideneimidazolinone compounds(BDI) was prepared and screened toward 94 biologically relevantanalytes to generate fluorescence response profiles. From theresponse pattern, compounds containing aminobenzyl and het-eroaromatic cyclic substructures revealed a pH dependent emis-sion decrease effect, and unlike other fluorescence scaffolds, mostBDIs showed fluorescence quenching when mixedwith proteins. On the basisof the primary responseprofile, we obtained three selectivefluorescence turn-on sensors for pH, human serum albumin (HSA), and total ribonucleic acid (RNA). Following analysis, a fluorescenceresponse profile testing four nucleic acids revealed the alkyloxy (Ph-OR) functional group in the para position of benzyl analoguescontributes to RNA selectivity. Among the primary hit compounds, BDI 2 showed outstanding selectivity toward total RNA with 5-foldemission enhancement. Finally, BDI 24 showed selective fluorescence increase to HSA (Kd = 3.57M) with a blue-shifted emission max

wavelength (em = 15 nm). Theseexamples offluorescencesensor discovery by large-scale fluorescence response profiling demonstratethe general applicability of this approach and the usefulness of the response profiles.

KEYWORDS: small molecule sensors, green fluorescent protein chromophore, fluorescence response profiling

INTRODUCTION

The green fluorescent protein (GFP) has attracted great atten-tion as a fluorescent probe to study diverse problems in molecularbiology.1 Taking advantage of the autocatalytic cyclization andoxidation of three amino acid residues to form a natural chromo-phore (p-hydroxybenzylideneimidazolone, p-HBDI),2 GFP gener-

ates a very eff

ective and intensefl

uorescence signal. Nevertheless,investigation of the chromophore reveals distinct differences influorescent properties between wild type GFP (wt GFP) and thesynthetic chromophore. The fluorescent quantum yield of thesynthetic chromophore is much less andshows solvent and temper-ature dependence.3 Studies suggest that the GFP-barrel structureplays a critical role in protecting the chromophore and reducingconformational flexibility and thus diminishing the rate of radiation-less decay. Within the rigid -barrel structure, p-HBDI undergoesexcited-state protontransfer(ESPT)4 from the chromophore to theadjacentresidue,E222,5 resulting in very efficient emission fromtheanionic form. To exploit the GFP chromophore as a sensor, thisenvironment-sensitive fluorescenceturn-on phenomenonis utilized

in several bimolecular fluorescence protein sensors.6 However, thechromophore itself is rarely scrutinized to detect analyte moleculesdirectly.

We envisioned that synthetic GFP chromophore derivativeshave superior potential as small molecule fluorescent sensors,because of their desirable characteristics such as extremely lowbackground in aqueous solution and high environmental sensi-tivity. Low background sensors are favorable in many applica-tions because of their high signal-to-noise ratio.7 Recently, theBurgess group has demonstrated that, by incorporating an ortho-aryl BF2 group which complexes with the imidazolone nitrogen,the conformation of the chromophore can be frozen and theemission turned on.8 Similarly, Chou has demonstrated thatincorporation of an ortho hydroxyl group can turn on theemission through intramolecular hydrogen bonding, leading inthe latter case to excited-state proton transfer.9 We concluded

Received: September 22, 2010
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33 dx.doi.org/10.1021/co100012k | ACS Comb. Sci. 2011, 13, 3238

ACS Combinatorial Science RESEARCH ARTICLE

that such fluorescent turn-on detection for target moleculescould be attained by specific interactions between GFP chromo-phores and analyte molecules, which instead of freezing theconformation intrinsically through the structure of the chromo-phore, instead freezes the conformation after complexation withthe analyte. Indeed, recently we observed that proper construc-tion of a hydrophobic chromophore could increase the fluores-

cence response by a factor of 15.10

Accordingly, it would be ofinterest to investigate the chromophore modes of interactionwith endogenous biomolecules, despite most previous studies,which focused on the nature of chromophore within the-barrelin wt GFP. In this paper, we report a systematic analysis of thefluorescence change of the GFP chromophore and its derivativeswhen applied for sensing of biologically relevant analytes.

To develop an effective fluorescent sensor, an importantcriterionthat must be met is high selectivity for a target molecule.The selectivity of probes can be evaluated by parallel comparisonagainst a similar class of analytes. For example, Chang's groupvalidated selectivity of their metal probes by carefully comparingvarious kinds of metal ions together with the target metal ions.11

Many examples of diversity-oriented fluorescence sensors were

discovered by in vitro fluorescence response assessments prior totheir application in a biological system.12,13 It is noteworthythat the fluorescence response profile against diverse analytescould provide ultimate selectivity information. While similarprofiling approaches have been widely used in drug discovery,14

their application to develop fluorescent sensors is still at an earlystage.

The challenge of large-scale, quantitative investigation offluo-rescence sensor development requires a practical high-throughputscreening platform. For this, a microplate assay platform is chosenbecause of its unique advantages, such as simple adoption for anykindof analytes,flexible throughput control, and the homogeneousinteraction between a sensor molecule and an analyte. To broadenthe scope offluorescent sensor discovery, we collected 10 classes of

bioanalytes (total 94 individual analytes) for high-throughput invitro screening and performed an assay in 384-well microplates.From the resulting profile, we identified fluorescent small moleculesensors for pH, human serum albumin (HSA), and total ribonu-cleic acid (RNA).

RESULTS AND DISCUSSION

Design of BDI Compounds. The endogenous GFP chromo-phore contains two aromatic moieties, a para-hydroxy benzene

and an imidazolinone ring linked by a methine group. On thebasis of these building block structures, 41 benzylideneimidazo-linone (BDI) derivatives were synthesized with substitutionpatterns highlighted in Table 1. Since a lot of benzaldehyde building blocks are commercially available, we could easilymaximize structural diversity of the R1 moiety. Each BDIcompound shares a common core moiety; however, each ana-

logue has distinct structures that may provide unique fluorescentturn-on phenomena by interacting with target molecules.Synthesis of the BDIs was carried out by a 2 3 cycloaddi-

tion of the corresponding aromatic Schiffbase with the imidate(Scheme 1c).15

Photophysical properties in solution. The spectroscopicproperties ofBDI compounds were examined and are summarizedin Table 1. As previously reported, most BDI compounds showvery low extinction coefficients and fluorescence quantum yields inethanol. Each of these compounds absorbs in the visible range, withmax(abs) between341 and440 nm andhas fluorescence emissionranging from 464 to 599 nm.

BDI compounds that consist of either 4-diethylamino (BDI 24)or 4-dimethylamino (BDI 25, 33, 35) exhibit significantly longer

absorption max wavelengths than other derivatives (abs = 71.7nm between the mean absorbance wavelength of BDI 24, 25, 33,and 35 compared to other compounds). The notable red shift ofthe absorbance indicates the elongated push-pull electronicconjugation from the aminobenzyl group.

High Throughput in Vitro Fluorescence Response Profil-ing toward Biorelevant Analytes. Prior to experimentation,the solubility of all compounds was validated in 100M concen-tration of HEPES buffer (20 mM HEPES with 1% of the DMSOstock solution at pH 7.4). Under these conditions, all 41 BDIcompounds avoidedprecipitation,and the absorbance/fluorescencespectraexhibited reproducible peak shape in spite of their extremelyweak extinction coefficients and low quantum yields.

To systematically investigate the fluorescence response profile

of BDI compounds, 10 classes of analytes related to variousbiological processes were screened. The analyte classes were(i) pH standard solutions, (ii) viscous buffer solution, (iii) nucleo-tides andnucleosides,(iv) nucleic acids, (v) peptides,(vi) proteins,(vii) metal cations, (vii) oxidation-reduction related molecules,(viii) pesticides, and (ix) miscellaneous analytes, totaling 94individual analyte molecules (Supporting Information, Table S2).Each analyte wastested at four serialconcentrations together with agiven concentration of BDI compounds to determine the doseresponse dependence on their endogenous concentration or

Scheme 1. Synthetic Scheme of BDI Compounds: (A) Synthesis of Aromatic SchiffBase, (B) Synthesis of Imidate, (C) Synthesisof BDIsa

a Reactions were carried out by combining 1equiv of Schiffbase and 1.1 equiv of the imidate under ambient conditions with magnetic stirring.15
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effective concentration, and these fluorescent emission intensitieswere compared with the value of chromophore itself in the buffersolution. Additionally, the entire concentration-response patternwasused for investigation of the selectivity among the different setsof analytes.

Fluorescence spectra were obtained using a monochromator-based fluorescence microplate reader not only to investigateemission spectra change, but also to increase throughput.16

Excitation wavelengths of individual BDI molecules were deter-mined based on their maximum wavelength of absorbance in

Table 1. Substitution Patterns and Spectroscopic Properties of BDI Compoundsa

BDI # R1 R2 abs (nm) em (nm) (M-1 cm-1)

1 4-OH Me 368 473 2,596 0.0060

2 4-OMe Me 367 490 12,961 0.0018

3 4-OAc Me 350 472 5,106 0.0045

4 4-CO2H Me 356 474 5,913 0.0085

5 4-OBz Me 368 476 9,611 0.0020

6 3,5-t-Bu/4-OH Me 373 471 8,888 0.0021

7 3-OH Me 365 469 1,857 0.0247

8 2-(N)/4-OH Me 359 481 3,805 0.0485

9 4-Me Me 348 471 7,105 0.0044

10 3-Me Me 348 468 5,499 0.0050

11 2-Me Me 349 472 5,630 0.0034

12 2,4-Me Me 352 472 5,630 0.003613 2,5-Me Me 357 478 4,360 0.0041

14 4-Et Me 353 478 6,733 0.0032

15 2-CF3 Me 341 490 3,826 0.0455

16 3-OMe Me 355 510 9,501 0.0041

17 2,3-OMe Me 352 579 6,251 0.0471

18 2,4,5-OMe Me 413 527 5,894 0.0111

19 2,5-OMe Me 396 548 3,799 0.3062

20 4-NO2 Me 376 599 4,869 0.2008

21 4-Cl Me 352 472 6,598 0.0060

22 2-F Me 351 475 6,299 0.0096

23 4-CN Me 361 475 5,498 0.2490

24 4-N(Et)2 Me 438 527 12,817 0.0065

25 4-N(Me)2 Me 430 527 13,529 0.0029

26 1-naphthyl Me 374 476 2,247 0.2080

27 2-naphthyl Me 369 483 9,026 0.0048

28 2-quinoline Me 372 476 3,784 0.0971

29 1-naphthyl Et 378 464 5,214 0.0039

30 4-OH n-Pr 369 476 10,783 0.0018

31 4-Me n-Pr 351 470 8,334 0.0034

32 4-i-Pr n-Pr 353 509 6,434 0.0039

33 4-N(Me)2 n-Pr 433 504 8,604 0.0739

34 4-OH n-Bu 371 476 10,739 0.0018

35 4-N(Me)2 n-Bu 440 505 10,063 0.0650

36 4-OH n-Pentyl 371 503 9,031 0.0019

37 4-OH C3H6N(Me)2 372 471 4,867 0.002938 4-OH C3H6N(Me)3 369 472 4,091 0.0035

39 4-OH n-C11H22OH 369 554 8,007 0.0020

40 4-OH n-C16 371 504 6,723 0.0020

41 3-OH n-C16 353 474 3,328 0.0112aAll data were measured in ethanol with the presence of 1% DMSO as co-solvent. The fluorescence quantum yield was determined using FITC as astandard (fl = 0.93 in 0.1 M NaOH).


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20 mM HEPES. It should be noted that maximum wavelength ofexcitation and emission used in vitro screening are different fromthe values in Table 1 because of the solvent effect. Fluorescenceemission spectra were measured for all BDI compounds and 94analytes at 4 concentrations. To facilitate the broad use of thisdata, a fluorescence response profile database was configured(Supporting Information, Figure S2). Primary screening resultsof total spectra were stored in the database, and changes to the

emission spectrum were visualized by a heat map method. A heatmap is a graphical representation of fluorescence fold changeusing two color codes to simplify complexity of data. Greenrepresents an increase of emission intensity, while red representsa decrease in emission intensity. In this two-color profile, thefluorescence response pattern against 94 analytes could be easilyevaluated. For instance, Figure 1b shows the in vitro fluorescenceresponse profile ofBDI 8, which showed strong pH dependencewith a fluorescence increase in acidic media as well as showing afluorescence increase in the presence of onemetal cationand twomiscellaneous analytes in a dose dependent manner. The con-centration dependent response pattern is useful to evaluatethe quality of an assay, and allows for the determination of

dynamic ranges and sensitivity of probes.17 For those analytesthat showed remarked response, further experiments were pur-sued to determine the sensitivity for selective sensor application.The given profiles were analyzed for structure fluorescenceresponse relationships.

Fluorescence Response Profile and Structural Relationship.The present profile allows for evaluation of the fluorescenceintensity change of a given BDI compound with its structuralbuilding blocks. In pH profiles, all amino benzyl containing BDIcompounds (BDI 24 and 25) revealed strong fluorescencequenching in acidic conditions, which most likely is induced byN-protonation (Figure 2a, pH=2

BDI 24 = 0.002, pH=7BDI 24 =

0.021). Compounds containing different functional groups in theortho position showed a distinct pH dependent pattern. Four func-tional moieties were compared within a given series of equivalentR2 substitutions including compounds of methyl (BDI 11, 12, 13),trifluoromethyl (BDI 15), fluoro- (BDI 22), and methoxy- (BDI17, 18, 19), with fluorescence quenching in acidic solution ob-served for only ortho-methoxy compounds (Figure 2b,c). Singleatom substitution in the aromatic ring also induced dramaticchanges in the fluorescence response patterns. The wt-GFP chro-mophore, BDI 1, showed slight emission enhancement in basicmedia; however, its pyridine derivative, BDI 8, exhibited a strongemission enhancement in acidic solution(Figure 2d,pH=2

BDI 8 =0.786, pH=7

BDI 8 =0.008). Likewise, we observed an emissiondecrease from the quinoline derivative (BDI 28), whereas the

Figure 1. (a) Structure ofBDI8, and (b) in vitro fluorescence responseprofile of BDI 8. The number inside the parentheses indicates the

number of individual analytes. Green color representsfl

uorescenceintensity increase, and red color represents fluorescence decrease asdenoted in the scale bar. Four serial concentrations were stacked in thesame column in a dose dependent manner. Fluorescence intensity foldchange was obtained at 520 nm (ex = 370 nm). For a detail decodingtable, refer to the Supporting Information, Table S3.

Figure 2. Heat map profiles of pH response from BDI compoundscontaining (a) para-amino benzyl, (b) ortho-methoxy moieties, and (c)other functional group in the ortho position. Fluorescence profilecomparison between homocyclic and heterocyclic rings; (d) p-hydroxyl

benzyl and p-hydroxyl pyridine derivatives, (e) naphthalene and quino-line derivatives.
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naphthalene derivative (BDI 29) showed minimal pH response(Figure 2e).

Similar structural analysis could also be performed based on thestructure of analytes. Within the miscellaneous analyte class,primary amines caused fluorescence enhancement at high con-centration for most BDI compounds (Supporting Information,Figure S3). Since the pH changes induced by two primary amines,

dopamine and histamine, were already included in the pH profi

le,it is believed that BDI compounds share common interactionmodes with those primary amines. Another notable response is thehydrophobic interaction with macromolecules. Manyfluorescentsmall molecules, including 8-aminonaphthalenesulfonamide orrosamine compounds, show non-specific fluorescence increaseto hydrophobic proteins such as albumin.13 Conversely, most BDIcompounds exhibited fluorescence quenching toward albumins.Considering the fluorescence enhancement in wt-GFP stimulatedby rigidifying the chromophore structure, this profile implies thatprotein analytes do not have a common binding pocket for theBDI scaffold.

The results of these analyses demonstrate that the profilesgenerated are useful to analyze the relationship between fluo-

rescence responses and structural characteristics in both analytesand probes. With this general response pattern information, wefurther sought to discover selective fluorescence turn-on sensors.

Fluorescence Sensor Discovery from the Profile. To bemaximally useful, the fluorescence response profile should allowfor the discovery of novel sensor molecules. Since we initiallyclassified analytes in terms of the nature of the individual analyte,it was straightforward to compare fluorescence response withinthe same class of analytes. In this study, we found two sensormolecules from separate analyte classes that warranted furtherinvestigation.

(i). RNA Sensors. Although fluorescence small molecule sen-sors for nucleic acid macromolecules have been extensively usedfor cell imaging and sequencing, the continued development of

improved sensors that can distinguish different kinds of nucleicacids is currently of great interest.18 Accordingly, 4 kinds ofnucleic acids were collected as a primary screening analyte classto discover sensors that could discriminate double strand DNA(dsDNA), single strand DNA (ssDNA), RNA, and t-RNA. Firstof all, we found BDI 37 and BDI 38 exhibited an increase influorescence emission for all four kinds of nucleic acids from theprimary screening (Supporting Information, Figure S4). BDI 38contain a positively charged linker in the BDI derivatives, and ithas strong Coulomb interaction with all negatively chargednucleic acids.

Primary hit compounds that enhanced emission in the pres-ence of RNA compared to DNA were further tested to confirmprimary profiles, and 3 BDI compounds revealed selectivity

toward total RNA. Interestingly, all selected RNA sensors, BDI2, BDI 3, and BDI 5, contain alkyloxy/aryloxy groups (-OR) atthe para position in the R1 moiety. These findings suggest thatthe para-alkyloxy group plays a role in distinguishing RNAs overDNAs in BDI scaffolds. We also note that thepara-alkoxy groupsplayeda significant role in enhancing thefluorescence in the solidstate.18 Among the three primary hits, the para-methoxy groupbrought not only the highest fold change in fluorescence to totalRNA, but also exceptionally low cross reactivity to other nucleicacids (Figure 3). When we tested these hit compounds towarddifferent 16-mer RNA oligomers, they generally exhibited higherfluorescence increment toward AU rich oligomers (SupportingInformation, Figure S6). Although we observed a response trend

against AU rich oligomers, it is still not clear which specificRNA sequences were necessary for selective recognition of eachcompound or how these compounds were interacting with totalRNA mixture in solution. However, this example clearly showsthat large-scale profiling could identify novel modes of interac-tion between sensor molecule and analyte. Such studies arecurrently in progress.

(ii). Human Serum Albumin (HSA) Sensors. As mentionedearlier, most BDI compounds showed fluorescence quenchingtoward many proteins (Supporting Information, Figure S7).Noticeable primary hit compounds were BDI 24 and BDI 25.

These two compounds, which contain a dialkylamino motif,showed clear emission enhancement to human serum albumin(HSA) in a dose dependent manner.

HSA is the most abundant protein in human blood plasma(60% of total plasma protein) and is known to bind to diverseexogenous drug molecules by hydrophobic interactions.19 SinceBDI 24 exhibited a greater fold increase, we further examined thebindings between HSA and BDI 24. In the presence of HSA, themaximum wavelength offluorescence emission was blue-shiftedfrom 530 to 515 nm, while the intensity increased by 10.4 fold(Figure 4a, Supporting Information, Figure S8). This hypso-chromic shift can be causedby solvatochromism, thereduction ofnon-radiative decay, or the perturbation of electronic conjuga-tion systemthrough the binding with HSA. To further investigate

the binding properties of HSAand BDI 24 quantitatively, variousconcentrations of BDI 24 were titrated with HSA, and a frac-tional saturation curve revealed a dissociation constant (Kd) of3.57 M (Figure 4b).

CONCLUSION

Although the sensitivities displayed here do not compare toothers for the same proteins, they are based upon a limited initialset of chromophores. Nevertheless, this study represents the firstexample of small moleculefluorescence sensor discovery utilizingthe green fluorescent protein chromophores by large-scalefluorescence profiling. We believe this profiling approach couldaccelerate the fluorescence sensor discovery process for other

Figure 3. Fluorescence intensity fold changes of (a) BDI 2 at em 512nm, (b) BDI 3, and (c) BDI 5 at em 532 nm against 4 nucleic acids at

1 mg/mL finalconcentrationsin 20 mM HEPES (pH7.4). Full emissionspectra (Supporting Information, Figure S5).


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fluorescent scaffolds, since the diversity of the fluorophoresintroduced here represents a very small fraction of the potentialstructural space. In the current report, we have demonstrated theuse of the fluorescence response pattern based sensor discoveryutilizing the green fluorescent protein chromophore and itsderivatives. Forty-one structurally related BDI compounds weresynthesized and exploited for the generation of fluorescenceresponse profiles toward 10 classes of analytes or a total of 94individual biologically relevant analytes. From the large-scalefluorescence profile, the structural moieties that induce specificpH response were identified as well as those showing a uniqueresponse pattern to specific analytes. Further experiments ex-hibited a selective fluorescence emission enhancement uponinteraction between BDI 24 with HSA (Kd = 3.57 M) and

BDI 2 with total RNA, which showed a marked selectivity with a5-fold emission increase. The use of more carefully focusedrecognition elements should increase the sensitivity and ap-proach the on/offratio of GFP itself. For instance, we now haveincreased the sensitivity to the hydrophobic octa acid byanother factor of 10.20 Further studies are currently in progress.

EXPERIMENTAL SECTION

Materials and Methods. All the materials were obtained fromcommercial suppliers (Aldrich) and used without further purifica-tion. The solvents used for the spectroscopy experiments were ofspectrophotometric grade. Spectroscopic properties of com-pounds and in vitro high-throughput screenings were performed

on SpectraMax M2 plate reader (Molecular Devices Inc.).Quantum Yield Measurements. Quantum yields were cal-

culated by measuring the integrated emission area of the fluo-rescent spectra andcomparing that value to thearea measured forFITC in EtOH when excited at 489 nm (FITC = 0.93). Quan-tum yields for the BDI compounds were then calculated usingeq 1, where F represents the area of fluorescent emission, n isreflective index of the solvent, and Abs is absorbance at excitationwavelength selected for standards and samples.

sample

flu reference

fl

Fsample

Freference

!sample

reference

!Absreference

Abssample

!1

In Vitro Fluorescence Screening. BDI compounds (100M) were screened in 20 mM HEPES, pH 7.4. Fluorescenceintensities were measured using a SpectraMax M2 plate readerin 384-well format. Excitation was provided at each com-pound's excitation range, and emission was obtained startingfrom at least 30 nm longer wavelengths from the excitation. Allthe analytes were tested at four serial concentrations depend-ing on the endogenous concentration of the analyte (Support-ing Information, Table S2). Every analyte was prepared thesame day prior to the fluorescence experiment to minimizesample contamination. For detailed protocols refer to theSupporting Information.

Determination of the Dissociation Constant. The fluores-cent emission spectra with various concentrations of BDI

compounds were measured on a SpectraMax M2 plate reader.The fluorescent titration curve was fitted to the standardequation using Graphpad Prism v5 software. The bound fraction(X) of fluorescent sensor at each concentration was determinedusing the eq 2

X Fc- Fo

Fsat- Fo2

where Fc and Fo are the fluorescence intensities of a givenconcentration of BDI compounds with and without targetanalyte, respectively. Fsat is the fluorescence intensity at thesame concentration of BDI compounds when fully bound with target analyte. Fsat was determined by fluorescencetitration at each concentration with a series concentration of

analyte. The results were plotted according to a non-linearfitting curve eq 3

F Fo Fsat- FoBDI=KD BDI 3

where KD is dissociation constant, and [BDI] is the concen-tration of the BDI compound.

ASSOCIATED CONTENT

bS Supporting Information. Detail characterizations of allBDI compounds, in vitro screening procedures/results, andcomplete refs 14a, 14d. This material is available free of chargevia the Internet at http://pubs.acs.org.

Figure 4. Fluorescence titration analysis for the binding ofBDI 24 with HSA. (a) Fluorescence spectral change ofBDI 24 (100 uM) upon addition ofthe HSA in 20 mM HEPES buffer (pH 7.4). (b) Fractional saturation curve ofBDI 24with HSA. The dissociation constant was measured using 7.5 MHSA. Experimental Kd = 3.57 M.


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AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected].

Author Contributions3These authors contributed equally to this work.

ACKNOWLEDGMENT

This work wassupportedby anintramural fundingfromA*STARBiomedical Research Council and National University of Singapore(Young Investigator Award: R-143-000-353-123). J.-S.L. thanks thePOSCO TJ Park Foundation (TJ Park Bessemer Science Fellow-ship) for the generous support. L.M.T. thanks the National ScienceFoundation through Grants CHE-0809179 for financial support.A.B. acknowledges the Center for Organic Photonics and Electron-ics (COPE) at Georgia Tech for a graduate research fellowship.

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